Developed defined methods to grow replicative cultures of normal human bronchial epithelial cells either without serum or Swiss mouse 3T3 feeder-cells. These cells can be subcultured several times, will undergo 35 population doublings and have the expected epithelial cell characteristics of keratin, desmosomes and blood group antigens. Further, mitotically quiescent cells will differentiate into cells with beating cilia, a characteristic of normal bronchial epithelium. Dissociated single cells form colonies when plated at low density. In vitro carcinogenesis experiments with normal bronchial epithelial tissue and cell cultures have yielded populations of cells which have abnormal characteristics. These phenotypically altered cells (PACS), which have the keratin epithelial cell markers, have extended population doubling potentials, abnormal human karyologies and abnormal serum and growth factor requirements. The tumorigenic potential of these isolates are being assessed in athymic nude mice.